The present invention relates to modifying the use of .alpha.-amylase in conversion of grain starch to downstream products, such as dextrose, fructose and alcohol. In particular, the present invention relates to the addition of sodium ion to a starch mixture prior to or during liquefaction to increase the efficiency of liquefaction at low pH, i.e., at or below about pH5.9.
Grains such as corn have long been used as a source of starch. One of the well known methods of separating and purifying starch for use in industrial processes is the wet-milling process. This method has developed into a highly specific and integrated system designed to separate the major components of a grain kernel as completely as possible (see Stanley A. Watson, Starch: Chemistry & Technology, Vol. II , Industrial Aspects, Academic Press, New York, 1967, pp. 30-51).
In a common wet-milling process, dry grains used for the production of starch products are first subjected to a soaking process called steeping. During steeping, the grains are subjected to a counterflow water current which separates many solubles, including phytate and phytic acid, sugars, salts and proteins, from the grain granules. The steeped grains are separated from the soaking water (steepwater) and subjected to mechanical cracking and grinding procedures. Flotation and centrifugation techniques are then used to separate germ from the starch, fiber and protein. The resulting slurry of endosperm (starch), fiber and protein is then further ground and screened to separate out the fiber. Finally, the protein and endosperm related components are separated based on density through countercurrent rinsing and centrifugation to separate the starch from the protein/gluten stream. The isolated starch stream is then extensively rinsed to remove any non-granular starch related solubles, including solubles such as inorganic salts, and compounds such as phytate and salts of phytic acid. The resulting product is a highly purified slurry of insoluble granular starch which serves as the starting product for conversion to fructose.
In general, starch to fructose processing consists of four steps: liquefaction of granular starch, saccharification of the liquefied starch into dextrose, purification, and isomerization to fructose. The object of a starch liquefaction process is to convert a concentrated suspension of starch polymer granules into a solution of soluble shorter chain length dextrins of low viscosity. This step is essential for convenient handling with standard equipment and for efficient conversion to glucose or other sugars. To liquefy granular starch, it is necessary to gelatinize the granules by raising the temperature of the granular starch to over about 72.degree. C. The heating process instantaneously disrupts the insoluble starch granules to produce a water soluble starch solution. The solubilized starch solution is then liquefied by .alpha.-amylase (EC 3.2.1.1.).
A common enzymatic liquefaction process involves adjusting the pH of a granular starch slurry to between 6.0 and 6.5, the pH optimum of .alpha.-amylase derived from Bacillus licheniformis, with the addition of calcium hydroxide, sodium hydroxide or sodium carbonate. The addition of calcium hydroxide has the advantage of also providing calcium ions which are known to stabilize the .alpha.-amylase against inactivation. Upon addition of .alpha.-amylase, the suspension is pumped through a steam jet to instantaneously raise the temperature to between 80.degree.-115.degree. C. The starch is immediately gelatinized and, due to the presence of .alpha.-amylase, depolymerized through random hydrolysis of a(1-4) glycosidic bonds by .alpha.-amylase to a fluid mass which is easily pumped.
In a second variation to the liquefaction process, .alpha.-amylase is added to the starch suspension, the suspension is held at a temperature of 80-100.degree. C. to partially hydrolyze the starch granules, and the partially hydrolyzed starch suspension is pumped through a jet at temperatures in excess of about 105.degree. C. to thoroughly gelatinize any remaining granular structure. After cooling the gelatinized starch, a second addition of .alpha.-amylase can be made to further hydrolyze the starch.
A third variation of this process is called the dry milling process. In dry milling, whole grain is ground and combined with water. The germ is optionally removed by flotation separation or equivalent techniques. The resulting mixture, which contains starch, fiber, protein and other components of the grain, is liquefied using .alpha.-amylase. The general practice in the art is to undertake enzymatic liquefaction at a lower temperature when using the dry milling process. Generally, low temperature liquefaction is believed to be less efficient than high temperature liquefaction in converting starch to soluble dextrins.
Typically, after gelatinization the starch solution is held at an elevated temperature in the presence of .alpha.-amylase until a DE of 10-20 is achieved, usually a period of 1-3 hours. Dextrose equivalent (DE) is the industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE of virtually zero, whereas the DE of D-glucose is defined as 100.
The maximum temperature at which the starch solution containing .alpha.-amylase can be held depends upon the microbial source from which the enzyme was attained and the molecular structure of the .alpha.-amylase molecule. .alpha.-amylases produced by wild-type strains of B. subtilis or B. amyloliquefaciens are typically used at temperatures no greater than about 90.degree. C. due to excessively rapid thermal inactivation above that temperature, whereas .alpha.-amylases produced by wild-type strains of B. licheniformis can be used at temperatures up to about 110.degree. C.
The presence of starch and calcium ion are known to stabilize .alpha.-amylases against inactivation. Nonetheless, .alpha.-amylases are used at pH values above 6 to protect against rapid inactivation. At low temperatures, .alpha.-amylase from B. licheniformis is known to display excellent hydrolyzing activity on starch substrate at pH values as low as 5. However, when the enzyme is used for starch hydrolysis at common jet temperatures, e.g., between 102.degree. C. and 109.degree. C., the pH must be maintained at least above pH 5.7 to avoid excessively rapid inactivation. The pH requirement unfortunately provides a narrow window of processing opportunity because pH values above 6.0 result in undesirable by-products, e.g., maltulose. Therefore, in reality, liquefaction pH must be maintained between 5.9 and 6.0 to attain a satisfactory yield of hydrolyzed starch.
Another problem relating to pH of liquefaction is the need to raise the pH of the starch suspension from about 4, the pH of a corn starch suspension as it comes from the wet milling stage, to 5.9-6.0. This pH adjustment requires the costly addition of acid neutralizing chemicals and also requires additional ion-exchange refining of the final starch conversion product to remove the chemical. Moreover, the next process step after liquefaction, typically saccharification of the liquefied starch into glucose, requires a pH of 4-4.5; therefore, the pH must be adjusted down from 5.9-6.0 to 4-4.5; requiring additional chemical addition and refining steps.
In U.S. Pat. No. 5,322,778, liquefaction between pH 4.0 and 6.0 was achieved by adding an antioxidant such as bisulfite or a salt thereof, ascorbic acid or a salt thereof, erythorbic acid, or phenolic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, or .alpha.-tocopherol to the liquefaction slurry. According to the claims of this patent, the antioxidant must be added in a concentration of greater than 5 mM.
In U.S. Pat. No. 5,180,669, liquefaction between a pH of 5.0 to 6.0 was achieved by the addition of carbonate ion in excess of the amount needed to buffer the solution to the ground starch slurry. Due to an increased pH effect which occurs with addition of carbonate ion, the slurry is generally neutralized by adding a source of hydrogen ion, for example, an inorganic acid such as hydrochloric acid or sulfuric acid. In PCT Publication No. WO 94/02597, a mutant .alpha.-amylase having improved oxidative stability is described wherein one or more methionines are replaced by any amino acid except cysteine or methionine.
In PCT publication No. 94/18314, a mutant -amylase having improved oxidative stability is described wherein one or more of the methionine, tryptophan, cysteine, histidine or tyrosine residues is replaced with a non-oxidizable amino acid.
In PCT Publication No. WO 91/00353, the problems associated with liquefaction are approached by genetically engineering .alpha.-amylase to include characteristics including increased thermal, acid and alkaline stability.
In U.S. Pat. No. 4,914,029, phytase is added to the corn steep liquor to reduce the quantity of phytic acid in the corn steep liquor, and thus more efficiently utilize the corn steep liquor in animal feed.
Despite the advances made in the prior art, a continuing need exists for efficient means for starch liquefaction at low pH levels using commercially available .alpha.-amylase.